In vivo fluorescence imaging, utilizing fluorescent reporter proteins, or exogenously administered fluorescent probes, plays an important role in the interrogation of biologic systems, particularly the study of rodent models of human disease. The majority of systems to date produce 2-D images of the integrated light distribution emitted at the surface of the animal, severely compromising the ability to quantify and accurately localize signals due to the strong depth dependence of the optical signal. There is great interest in extending in vivo fluorescence imaging methodology to the reconstruction of volumetric images that can show the location and concentration of fluorescent proteins or fluorescent contrast agents in living mice. This would be significant, as it would enable quantitative molecular imaging with fluorescent approaches at considerably less cost and potentially higher spatial and temporal resolution than competing modalities such as positron emissiontomography. Incorporating spectral information into the reconstructionprocess provides additional information about source depth due to the dependence of tissue absorption and scattering properties on wavelength. Spectral information therefore has the potential to increase depth resolution of fluorescence reconstruction as compared to reconstruction from traditional monochromatic or broadband measurements. Furthermore, by using a whole camera image instead of a small number of optical detectors for reconstruction, the amount of information gathered from the surface of the animal is increased and should also contribute to enhanced fluorophore reconstructions. For the proposed project, we plan to develop a hyperspectral optical imaging system and algorithms, which are capable of quantitative three-dimensional tomographic reconstructions of fluorophore distributions in small animals. In order to achieve this goal, a number of critical research issues will be addressed, including system design, optimization of excitation and detection geometries, efficient and accurate modeling of light transport in the mouse and efficient image reconstruction strategies. A series of simulations, phantom and animal experiments will be used to validate the approach and the results will be compared to standard broadband methods that utilize high-pass filters.